Patient-specific retinal organoids demonstrate the sound nature of Knudson's hypothesis

What is it about?

Retinoblastoma (Rb) is a type of malignant tumor due to abnormal retinogenesis with biallelic mutations of the RB1 gene. Its pathogenesis has been proposed as a “two-mutation hypothesis” by Knudson since 1971; however, there remain some debates on disease onset sufficiency of the biallelic RB1 mutations. To obtain straightforward evidence for this hypothesis, we investigated that whether two-hit mutations of the RB1 gene drive tumorigenesis in a patient induced pluripotent stem cells (hiPSCs)-derived human retinal organoids (hROs) and that whether single allelic mutation hiPSCs-derived hROs exhibit molecular and cellular defects. We generated hiPSCs with a heterozygous germline mutation (RB1m1/wt) from a Rb patient. A second-allele RB1 gene mutation was knocked in to produce compound heterozygous mutations (RB1m1/m2) in the hiPSCs. These two hiPSC lines were independently developed into hROs through a stepwise differentiation. The hiPSC-RB1m1/m2 derived organoids demonstrated tumorigenesis in dishes, consistent with Rb profiles in spatiotemporal transcriptomes, in which developmentally photoreceptor fate-determining markers, CRX and OTX2, were highly expressed in hiPSC-RB1m1/m2 derived hROs. Additionally, ARR3+ maturing cone precursors were co-labeled with proliferative markers Ki67 or PCNA, in agreement with the consensus that human Rb is originated from maturing cone precursors. Finally, we demonstrated that retinal cells of hROs with monoallelic RB1 mutation were abnormal in molecular aspects due to its haploinsufficiency. In conclusion, this study provides straightforward supporting evidence in a way of reverse genetics for “two-hit hypothesis” in the Rb tumorigenesis and opens new avenues for development of early intervention and treatment of Rb.

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Photo by Harpreet Singh on Unsplash

Photo by Harpreet Singh on Unsplash

Why is it important?

According to Knudson’s “two-hit” hypothesis, biallelic RB1 mutations are critical for Retinoblastoma (Rb) formation, but there is no biological model to test this hypothesis in a straightforward way. Here, we constructed Rb organoids derived from patient-iPSCs with a germline “hit” in the RB1 gene and an extra-mutation knocked in by CRISPR/Cas9. Under stepwise differentiation, the Rb organoids were successfully formed in petri dishes with typic tumor signatures and excessive proliferation of cone precursors. In addition, although the patient-iPSCs with a monoallelic mutation did not produce Rb, significant alterations in molecular aspects presented in the retinal organoids due to the haploinsufficiency. In brief, we established Rb organoid model with patient specific genetic background and provided straightforward evidence testifying Knudson’s theory.

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